Real-time quantitative PCR assays for evaluation of soybean varieties for resistance to the stem and root rot pathogen Phytophthora sojae

Abstract

Phytophthora root and stem rot caused by Phytophthora sojae is one of the most destructive disease of soybeans in the world. Effective management of the disease depends on selection and use of soybean varieties resistant to the disease. Fast and reliable procedures are vital to screen soybean varieties against the pathogen. Novel real-time quantitative (qPCR) assays were developed for both absolute and relative quantification of P. sojae in infected root tissues. QPCR assays were based on the detection of the internal transcribed spacer (ITS) gene of the pathogen and 18S ribosomal gene of the host plant. Absolute qPCR allowed the detection of as low as 10 femtograms (fg) of P. sojae DNA in soybean roots. Relative qPCR, employing the comparative threshold cycle (Ct) method, was effective and reliable for quantification of P. sojae DNA normalized to plant DNA in infected soybean root tissues. P. sojae DNA quantities detected in both qPCR assays had high correlations with disease severity index (DSI) ratings of soybean varieties. QPCR assays developed in this study were useful for determination of the levels of P. sojae DNA in different varieties of soybean and for evaluation of them for relative resistance to the pathogen.